Streptolysin O Deficiency in Streptococcus pyogenes M1T1 covR/S Mutant Strain Attenuates Virulence in In Vitro and In Vivo Infection Models

ABSTRACT Mutation within the Streptococcus pyogenes (Streptococcus group A; Strep A) covR/S regulatory system has been associated with a hypervirulent phenotype resulting from the upregulation of several virulence factors, including the pore-forming toxin, streptolysin O (SLO). In this study, we utilized a range of covR/S mutants, including M1T1 clonal strains (5448 and a covS mutant generated through mouse passage designated 5448AP), to investigate the contribution of SLO to the pathogenesis of covR/S mutant Strep A disease. Up-regulation of slo in 5448AP resulted in increased SLO-mediated hemolysis, decreased dendritic cell (DC) viability post coculture with Strep A, and increased production of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) by DCs. Mouse passage of an isogenic 5448 slo-deletion mutant resulted in recovery of several covR/S mutants within the 5448Δslo background. Passage also introduced mutations in non-covR/S genes, but these were considered to have no impact on virulence. Although slo-deficient mutants exhibited the characteristic covR/S-controlled virulence factor upregulation, these mutants caused increased DC viability with reduced inflammatory cytokine production by infected DCs. In vivo, slo expression correlated with decreased DC numbers in infected murine skin and significant bacteremia by 3 days postinfection, with severe pathology at the infection site. Conversely, the absence of slo in the infecting strain (covR/S mutant or wild-type) resulted in detection of DCs in the skin and attenuated virulence in a murine model of pyoderma. slo-sufficient and -deficient covR/S mutants were susceptible to immune clearance mediated by a combination vaccine consisting of a conserved M protein peptide and a peptide from the CXC chemokine protease SpyCEP.

IMPORTANCE Streptococcus pyogenes is responsible for significant numbers of invasive and noninvasive infections which cause significant morbidity and mortality globally. Strep A isolates with mutations in the covR/S system display greater propensity to cause severe invasive diseases, which are responsible for more than 163,000 deaths each year. This is due to the upregulation of virulence factors, including the pore-forming toxin streptolysin O. Utilizing covR/S and slo-knockout mutants, we investigated the role of SLO in virulence. We found that SLO alters interactions with host cell populations and increases Strep A viability at sterile sites of the host, such as the blood, and that its absence results in significantly less virulence. This work underscores the importance of SLO in Strep A virulence while highlighting the complex nature of Strep A pathogenesis. This improved insight into host-pathogen interactions will enable a better understanding of host immune evasion mechanisms and inform streptococcal vaccine development programs.
T he exclusively human pathogen Streptococcus pyogenes (Streptococcus group A-as above; Strep A) causes a variety of illnesses ranging from mild and self-limiting infections such as 'strep throat' to invasive life-threatening diseases, including necrotizing fasciitis, which commonly commences with a skin infection. Additionally, the development of post-streptococcal sequelae such as acute rheumatic fever (ARF) and rheumatic heart disease (RHD) contribute significantly to high rates of morbidity and mortality in developing countries and among Indigenous people of developed nations. The burden of ARF and RHD is particularly high in the Indigenous population of Australia, with prevalence rates of RHD reportedly up to 32 cases per 1,000 people (1,2). Globally, invasive streptococcal diseases and post-streptococcal sequelae are reported to contribute to more than 500,000 deaths annually, with many regions potentially underestimating Strep A-associated deaths (1,3).
The Strep A M1T1 clone is associated with an increased prevalence of invasive Strep A disease (4). The hypervirulence of this strain is suggested to be due to the ability of this clone to 'switch' to an invasive covR/S mutant phenotype (5)(6)(7)(8). The covR/S operon is a two-component negative transduction system that regulates between 10% and 15% of the streptococcal genome (9,10), the majority of which is involved in virulence factor expression and regulation. When the covR/S system acquires a spontaneous mutation, the resultant phenotype is hypervirulent and highly invasive in the host (5-8, 10, 11). This is clearly evidenced in the case of the invasive disease streptococcal toxic shock syndrome (STSS), where studies utilizing genome sequencing have shown that Strep A isolates possessing a mutation within covR/S were detected at a frequency of .50% in STSS clinical isolates, but only in 2% of noninvasive isolates (12,13).
The hypervirulence of covR/S mutants may be due to the de-repression of several virulence factors, each of which plays a critical role in immune evasion mechanisms employed by these strains (6). One of these critical virulence factors is streptolysin O (SLO). SLO, a potent pore-forming exotoxin, is a member of the cholesterol-dependent cytolysin family, which includes more than 28 members spanning several bacterial families (14). SLO primarily functions to induce pore formation in target host cells and inhibit phagocytosis, making it an important contributor to Strep A virulence (12,(15)(16)(17). Furthermore, the clinical relevance of SLO is underscored by the observation that SLO is commonly overproduced in STSS clinical isolates, which can lead to mortality rates as high as 30% to 70% depending upon the patient's geographical location and timely access to adequate health care (13,18).
Host defenses against Strep A infection are initiated by many immune cell types, including dendritic cells (DCs), which are prevalent in the skin and, together with neutrophils, constitute one of the first lines of defense. Strep A have developed several immune evasion mechanisms, including the ability to induce apoptosis of host cells. SLO significantly contributes to this process (15). Given that covR/S mutants have a propensity to initiate invasive infections, it might be hypothesized that upregulation of SLO by covR/S mutants plays a significant role in this hypervirulent phenotype.
Currently, there is no licensed Strep A vaccine; however, some experimental vaccines are showing promise and are at various stages of preclinical development (19)(20)(21)(22)(23). Developing a greater understanding of the complex interactions between host cells and Strep A can only contribute to hastening vaccine development. Such investigations have recently led to the redesign of an existing vaccine candidate, J8-DT/Alum, to provide greater protection against hypervirulent covR/S mutant Strep A (11,24). Immunization with the J8 peptide, which originates from the conserved region of the M protein, conjugated to diphtheria toxoid (DT) as a carrier protein, raises protective opsonic antibodies in vivo and is effective in several Strep A challenge models (19,25). However, the addition of a SpyCEP epitope (S2) to the J8-DT vaccine (herein called J8CombiVax) provides a synergistic protective response better equipped to afford protection against hypervirulent covR/S mutant infections (24). This was shown to work by inducing antibodies to negate the streptococcal CXC chemokine-degrading protease, SpyCEP, thus allowing anti-M protein antibodies to interact with neutrophils to kill the covR/S mutant Strep A.
With the goal of further understanding the pathogenesis of Strep A, we investigated the relative contribution of SLO in the context of covR/S gene regulation. Several different covR/S mutant Strep A isolates lacking the SLO gene (slo) were evaluated in vitro and in vivo using a murine model of pyoderma. Our data show that SLO is an essential factor for covR/S mutant-mediated hypervirulence.

RESULTS
Infection with Strep A 5448AP alters dendritic cell maturation and viability. DCs are important mediators of innate and adaptive immunity. Their importance in the context of Strep A infections in vitro (16) and following in vivo infection (26,27) has been previously demonstrated. Studies were undertaken to assess their relative contribution in a pyoderma model, where skin abrasion is followed by Strep A inoculation onto the lesion. Naive mice were infected with wild-type M1T1 5448 or covR/S mutant M1T1 5448AP, which are known to display differing virulence profiles (7,8,28). Skin sections were analyzed via immunohistochemistry (IHC) on day 3 postinfection. Staining the skin sections for CD207 highlighted a lack of skin-specific DCs (Langerhans cells) postinfection with 5448AP ( Fig. 1A) compared to postinfection with 5448 ( Fig. 1B) or naive skin (Fig. 1C). Quantification of CD207 1 DCs (via analysis of five separate high-powered fields) on day 3 postinfection demonstrated significantly fewer (P , 0.01) DCs in 5448AP-infected skin compared to 5448-infected skin (86% reduction) (Fig. 1D). This observation suggested that the maturation and/or viability of DCs was affected during 5448AP infection. To confirm this observation, DCs from a murine dendritic cell line, DC2.4, were infected with 5448 or 5448AP for 12 h in vitro. The subsequent expression of maturation markers (MHC II and co-stimulatory markers CD80 and CD86) was measured via flow cytometry. DC viability postinfection was also assessed using a live/dead stain. DC infection with 5448AP resulted in significantly reduced expression of MHC II, CD80, and CD86 in comparison to infection with 5448 (P , 0.05 to 0.01) ( Fig. 1E to G, respectively). Infection with either 5448AP or 5448 strains resulted in significantly higher DC death compared to the medium-only control; however, the viability of 5448AP-infected DCs was significantly less than that of 5448-infected DCs. (Fig. 1H). These data show that infection with 5448AP not only results in significantly fewer DCs in the skin but also induces a compromised maturation response in the remaining DCs.
SLO expression by covR/S mutant Strep A strains augments virulence. To investigate the mechanism by which 5448AP might be causing detrimental effects on DC maturation and survival, we first investigated the virulence profile of covR/S mutant isolates. A panel of four Strep A isolates and isogenic covR/S mutants (representing three emm types) was investigated in parallel (Fig. S1A). Reverse transcription-PCR (RT-PCR) was used to assess the relative expression of specific genes (sda1, speB, slo, cepA, hasA) during mid-log-phase growth. The wild-type (WT) expression of each gene for every isolate was standardized to a value of 1 relative to the housekeeping gene gyrase A, which was used as an internal control. All covR/S mutants demonstrated upregulated mRNA expression of slo, albeit at varying degrees, reflecting the inherent phenotypic differences between strains ( Fig. 2A). Expression of slo was most strongly upregulated in 5448AP, similar to previous reports in which slo is upregulated in a covR/S mutant background (4,6). We also assessed the functional changes in a SLO-mediated red blood cell (RBC) lysis assay using the 5448 and 5448AP strains. Culture supernatants from 5448AP were found to have significantly greater SLO-mediated hemolytic activity compared to supernatants from 5448 (Fig. 2B), which significantly correlated (P , 0.01) with the level of slo gene expression (Fig. S1B).
We next investigated whether other virulence factors were upregulated in 5448AP. RT-PCR was used to assess the expression of five important virulence genes under the control of covR/S in 5448 and 5448AP. We found that the RNA transcripts for SLO (slo), SpyCEP (cepA), and the hyaluronic acid capsule (hasA) were significantly upregulated in 5448AP compared to 5448 (P , 0.005 to P , 0.001) (Fig. 2C), similar to previous studies (6,8,28,29). This suggested that the enhanced virulence of 5448AP could be a cumulative effect of several virulence factors working in tandem. To further assess SLO- Contribution of SLO to covR/S Mutant Strep A Virulence mBio mediated effects in isolation, the chromosomal slo knockout strain 5448Dslo strain was employed. slo-deficient 5448 covR/S mutants demonstrate attenuated virulence. To investigate the role of SLO in the context of covR/S mutations, 5448Dslo was passaged through BALB/c mice to select for the covR/S mutant phenotype. After the fifth passage, the strain was considered mouse-adapted. From passage five, four individual colonies recovered from spleens were chosen for further characterization. Following sequencing of the covR/S operon, all four colonies were identified as covR/S mutants with mutations occurring at different locations within the operon (Table S1). 5448Dslo covR1 possessed full-length covR and covS genes with only a substitution mutation at nucleotide (nt) 341 of covR. In contrast, the three remaining mutants all possessed mutations within the covS gene, each resulting in a premature STOP codon. 5448Dslo covS mutants S1, S2, and S3 (designated 5448Dslo covS1, covS2, and covS3) possessed a truncated covS gene at nt 473, 199, and 9, respectively.
Each of the 5448Dslo covR/S mutants (covR1 and covS1-S3), as well as the covR/S WT parent 5448, 5448AP and 5448Dslo, were assessed for expression of covR/S-related virulence factors as described previously. The 5448AP covR/S mutant had significantly greater expression of cepA and hasA transcripts compared to 5448 (P , 0.001) (Fig. 3A). Passaging of 5448Dslo induced covR/S-mediated gene expression consistent with that of 5448AP (with upregulated cepA and hasA, and downregulated speB) (Fig. 3B). The 5448Dslo covS1 strain was most similar to 5448AP in terms of its gene expression profile and covS mutation location. Subsequently, 5448Dslo covS1 was chosen to undertake in vivo studies in mice. The gene expression profile of 5448Dslo was not significantly different from that of 5448 except for slo expression. 5448Dslo (and all derivatives generated) possessed no SLO-mediated hemolytic activity in vitro (Fig. S1C).
To examine broader genomic changes that may have occurred during animal passage, we performed genome sequencing on 5448, 5448AP, 5448Dslo, and 5448Dslo covS1. Comparative genomics of the four sequenced strains revealed maintenance of overall gene content with the 5448 reference strain (Fig. S2A). One exception was confirmation of the replacement of the slo gene with the cat resistance gene in the 5448Dslo genomic backgrounds (Fig. S2B). Single-nucleotide polymorphisms (SNP) and indel analysis revealed several SNPs across the genome of the four sequenced strains relative to the 5448 reference genome (CP008776). Two terminating insertions were identified in covS following Contribution of SLO to covR/S Mutant Strep A Virulence mBio animal passage: a single nucleotide insertion in the animal-passaged 5448AP strain and a two base-pair insertion in the 5448Dslo covS1 background. An additional non-synonymous SNP leading to the premature STOP codon was present in the type I restriction endonuclease gene hsdM (SP5448_08275) within the 5448Dslo genetic backgrounds (Table S2). hsd   (30,31). The virulence of slo-deficient 5448 and covR/S mutant strains was further assessed utilizing the DC2.4 murine cell line. DCs were infected in vitro with each of the 5448 variants for 12 h, and cell viability was assessed with flow cytometry using a live/dead stain. A comparison of DC viability between isogenic 5448 and 5448Dslo demonstrated significantly higher numbers of live DCs when they were infected with 5448Dslo (P , 0.05) (Fig. 3C). Similarly, DCs infected with the passaged slo knockout covR/S mutants were significantly more viable (P , 0.01 to P , 0.001) than DCs infected with slo-sufficient covR/S mutants (Fig. 3D). In all cases, the presence of slo was shown to play a critical role in the virulence mechanism of covR/S mutant strains.
To assess whether there was a difference in pro-inflammatory cytokine abundance in the supernatants of infected DCs, we utilized a cytokine bead array. The cytokine production was normalized to the same number of viable DCs across all cohorts. We found significantly greater amounts of tumor necrosis factor (TNF) and monocyte chemoattractant protein 1 (MCP-1) ( Fig. 3E and F, respectively) produced by DCs infected with 5448 and 5448AP (both expressing slo) in comparison to their slo-deficient derivatives (P , 0.05). Significantly higher production of TNF and MCP-1 (P , 0.05 to 0.001) by 5448AP-infected DCs was consistent with the SLO expression levels and activity seen with 5448AP ( Fig. 2A and B). Furthermore, these data demonstrated a role of SLO, in a covR/S mutant phenotype, in the modulation of pro-inflammatory cytokine responses.
Next, to assess the in vivo effect of SLO on the skin-resident DCs in mice, we performed IHC analyses. Naive BALB/c mice were infected with 5448, 5448AP, 5448Dslo, or 5448Dslo covS1 via the superficial skin infection method and skin samples were assessed on day 3. Although DC presence was evident in both 5448Dslo and 5448Dslo covS1-infected cohorts ( Fig. S3A and B), the numbers of DCs in both slo-deficient cohorts were significantly greater compared to those in 5448AP-infected skin (P , 0.005). These observations were consistent with in vitro data ( Fig. 3C and D) in which SLO-deficient mutants were shown to be significantly less detrimental to the survival of DCs. These data highlighted that SLO plays an important role in DC viability at the skin infection site. There was no significant difference in the number of DCs in 5448Dslo-infected or 5448Dslo covS1-infected skin (Fig. S3C), further implying SLO-mediated virulence mechanisms in skin infection.
To assess the in vivo virulence capacity of the slo-sufficient and -deficient covR/S mutants, naive BALB/c mice were infected with 5448AP or 5448Dslo covS1 using the superficial skin infection method (11). The skin biopsy specimens taken at day 3 postinfection showed that mice infected with the slo-sufficient strain 5448AP had more severe pathology at the infection site compared to mice with slo-deficient infection ( Fig. S4A and B, respectively). Both cohorts demonstrated comparable skin Strep A burdens on day 3 (Fig. 4A). However, by day 6, the Strep A burden in the skin of mice infected with the 5448Dslo covS1 strain was significantly less than that seen in mice infected with the slo-sufficient 5448AP (P , 0.05) (Fig. 4B). Furthermore, a significantly enhanced invasive ability of the slo-sufficient covR/S mutant 5448AP was evident in the blood (Fig. 4C and D) and spleen ( Fig. 4E and F) samples taken on days 3 and 6 postinfection.
Evasion of neutrophil killing is independent of SLO. The ability of each 5448 isolate to evade killing by human neutrophils was assessed. Neutrophils were isolated from three healthy volunteers and incubated with Strep A at an MOI of 1:10 (Strep A: neutrophils). Following an incubation period of 1 h, bacterial survival in the presence of human neutrophils was assessed.
The data demonstrated that at the MOI of 0.1, each of the three donors' neutrophils killed 5448 and 5448Dslo to a similar degree (survival ranged from 50% to 70%) (Fig. 5A). Both covR/S mutant organisms (5448AP and 5448Dslo covS1) grew significantly better in the presence of neutrophils compared to their corresponding WT isolates (P , 0.01 and 0.001, respectively). Notably, a lack of SLO on a covR/S phenotype (5448Dslo covS1) led to significantly higher evasion of neutrophil killing compared to that of a SLO-sufficient isolate (5448AP).
We observed an inverse correlation between Strep A survival and residual interleukin (IL)-8 produced by the neutrophils. A significantly greater amount of IL-8 was detected when the neutrophils were infected with 5448 compared to when they were infected with 5448AP (P , 0.01) (Fig. 5B), consistent with the upregulation of SpyCEP mRNA by 5448AP. This was also significantly higher than that in 5448Dslo covS1 (P , 0.05). IL-8 degradation caused by both the slo-deficient and -sufficient 5448 covR/S mutants was comparable (Fig. 5B). This result, and data showing that IL-8 degradation was similar in slo-sufficient and -deficient WT Strep A, suggests that the enhanced growth of SLO-deficient covR/S Strep A in the presence of human neutrophils is not linked to the strain's ability to degrade IL-8.
Vaccination with J8CombiVax compensates for neutrophil paucity and protects against covR/S mutant skin infection. Neutrophils and DCs are both critical for the adequate control of Strep A skin infection (11,12,16). Our in vitro and in vivo data suggested that SLO is responsible for an altered virulence of Strep A strains. Therefore, to Contribution of SLO to covR/S Mutant Strep A Virulence mBio assess the role of SLO in vaccine-mediated immunity, we performed skin challenges in which the protective efficacy of J8CombiVax (J8-DT1K4S2-DT/Alum) was assessed against slo-sufficient and -deficient 5448 covR/S mutants using the murine model of skin infection. BALB/c mice were immunized with J8CombiVax, then infected with 2 Â 10 6 CFU of 5448AP or 5448Dslo covS1 via the superficial skin infection method. On day 3 postinfection the vaccinated mice infected with slo-sufficient 5448AP or slo-deficient 5448Dslo covS1 both had significantly reduced skin Strep A burdens compared to their corresponding non-vaccinated controls (Fig. 6A). The mice infected with the 5448Dslo covS1 strain had significantly smaller lesions at the infection site compared to mice infected with the slo-sufficient covR/S mutant 5448AP, irrespective of immunization status (P , 0.05 to 0.005) (Fig. S4C). By day 6 postinfection, vaccinated mice challenged with 5448Dslo covS1 demonstrated a significantly greater reduction in skin Strep A burden compared to vaccinated mice challenged with the slo-sufficient 5448AP (P , 0.05) (Fig. 6B). There was no significant difference in lesion size at day 6 postinfection between all infected cohorts, although there was a trend of smaller lesions in mice challenged with slo-deficient Strep A (Fig. S4D). Strep A was not found in the blood or spleen of vaccinated mice following challenge with either 5448AP or 5448Dslo covS1 (Fig. 6C to F). The vaccine provided complete systemic protection against both isolates. However, in non-vaccinated mice, a significantly lower Strep A burden (P , 0.01) was noted for 5448Dslo covS1 in comparison to 5448AP, further demonstrating the attenuated invasive capability of slo-deficient 5448AP (Fig. 6C to F). Taken together, these data demonstrate that the altered virulence of slo-deficient 5448Dslo covS1 neither compromised nor augmented the efficacy of J8CombiVax against systemic infection.

DISCUSSION
Strep A covR/S mutants have an increased invasive propensity due to their ability to circumvent the host immune response and invade deep tissue sites (7,32). The hypervirulence of these strains contributes to severe Strep A infections worldwide. M1T1 strains isolated from invasive infections are often associated with covR/S mutation acquisition, and M1T1 is also overrepresented in invasive disease isolates (4, 32, 33). Understanding the complexities of the host-pathogen interaction is vital to the DCs are among the first responders at the infection site and thereby represent a crucial part of the innate immune response against streptococcal infections. A previous study highlighted the importance of DCs in immune responses against Strep A infection, with effective DC maturation being essential to this process (26). In this study, we observed that M1T1 5448AP induced less maturation of DCs in vitro than its covR/S WT counterpart, as indicated by the downregulation of MHC II, CD80, and CD86. CD86 is regarded as a marker of early DC maturation, with implications in initiation of immune responses by T cells; whereas CD80, being expressed on fully mature DCs, may function to amplify the immune response once it is initiated (34,35). A study by Borriello et al. (34) demonstrated that mice deficient in CD86 expression were greatly limited in their ability to induce a T-helper response and also presented a more severe immunodeficient phenotype. Not only do appropriate T cell responses enable the induction of high-affinity antibodies and antibody-producing B cells, but they also provide survival signals for the maintenance of memory B cells (36)(37)(38). Cytokines produced by T cells are responsible for the class-switching recombination event that distinguishes different immunoglobulin classes (38). In conclusion, appropriate and timely DC and T cell responses are essential for effective host immunity against Strep A.
Here, we hypothesized that the upregulated expression of slo by the covR/S mutant 5448AP was primarily responsible for this altered DC response. The murine histology data supported this notion, with slo-deficient 5448Dslo covS1 not inducing the same level of DC death at the infection site compared to the slo-sufficient 5448AP strain. This validates other studies in which Strep A isolates expressing high levels of SLO induced decreased maturation and apoptosis (as defined by the presence of hypodiploid nuclei) of DCs (16). Cortes and Wessels (16) also found that slo-expressing Strep A induced greater caspase production by infected DCs compared to slo-deficient strains. In our studies, we saw an upregulation of TNF by DCs. It is possible that 5448AP induces DC apoptosis via SLO-mediated TNF production which consequently leads to caspase activation and, ultimately, to DC death.
In several studies, slo expression has been linked to the impairment of phagocytes (12,15,17). However, in this study, we show that slo expression is important in the context of covR/S mutations for cellular interactions, potentially in partnership with the hyaluronic acid (HA) capsule. Several studies have highlighted a relationship between these two virulence factors, with its effects implicated in increased LL-37-induced resistance to killing by human cells (39) and altered cellular maturation responses (16), among others.
The relationship between SLO and the HA capsule can potentially be seen in the neutrophil interaction studies in which the covR/S mutants lacking slo (with significant upregulation of hasA compared to 5448Dslo) survived better in the presence of human neutrophils. This is in comparison to 5448Dslo (no significant difference in hasA gene expression compared to 5448), which was effectively killed by human neutrophils. The increased capsule production is likely responsible for the ineffective neutrophil killing in the absence of slo. Future studies would benefit from investigating a covR/S mutant double-knockout with slo and hasA. Furthermore, we observed a significantly greater amount of neutrophil-derived IL-8 produced after neutrophils were infected with 5448 compared to that after infection with 5448AP. This might be attributed to the significant increase in SpyCEP gene expression (cepA) by 5448 upon acquisition of the covR/S mutation, as seen in our gene expression studies and the findings of previous studies (40).
In addition to the altered in vitro responses, the 5448Dslo covS1 strain also demonstrated a diminished ability to disseminate from a superficial skin wound to cause systemic infections in a murine model. Furthermore, 5448Dslo covS1 produced less severe wounds at the infection site and significantly less bacteremia. This suggests that Strep A requires slo to invade and subsequently survive within sterile sites of the host, such as the blood. This observation is supported by a study by Zhu et al. (17) that highlighted the inability of slo mutants to cause cytotoxicity in keratinocytes. In the present study, we observed that the 5448Dslo covS1 strain resisted phagocytosis by human neutrophils in vitro but exhibited less virulence in the murine model of skin infection. This could be attributed to the inherent differences in cellular interactions in vivo compared to those modeled in vitro, where the intricate host immune systems are difficult to replicate. In addition, the differential behavior of mouse-adapted Strep A strains with human and mouse immune systems may also be contributing to this outcome. While these in vitro interaction studies are valuable for providing indications of virulence mechanisms, the utilization of murine models generally provides a more accurate depiction of host infections. Although the genomic sequencing confirmed that none of the observed differences between the SLO-deficient covR/S wild-type and mutant strain would have impacted the observed phenotype, it is possible that differential expression of other virulence factors may be contributing to overall virulence. Nevertheless, vaccination with J8CombiVax was able to effectively protect mice from infection with slo knockout strains despite their altered virulence (exhibiting upregulation of several covR/S mutation-mediated virulence factors).
One caveat of our study was the use of non-isogenic Dslo covR/S WT and covR/S mutant isolates (5448Dslo and 5448Dslo covS1). covR/S mutation in 5448Dslo was achieved through animal passaging, and it was not clear whether additional mutations were accrued during passage or whether they were manifested in the phenotypes observed. Whole-genome sequencing (WGS) addressed these concerns, confirming that the phenotypes observed in this study can be attributed to the slo gene and not to mutations in other genes introduced through animal passage. WGS of the strains investigated in this study identified expected mutations in the covS gene and an additional mutation of note to the hsdM gene. In covS (SP5448 08090), two terminating insertions were identified following animal passage: a single nucleotide insertion in the 5448AP strain (position 1,543,451 in 5448) and a two base-pair insertion in 5448Dslo covS1 (position 1,543,856 in 5448). As has been demonstrated in previous studies, passaged derivatives of a parent strain can exhibit multiple different covS mutations, including amino acid substitution and truncations, and these mutations have been shown to be responsible for virulence modulation (6,7).
An additional non-synonymous SNP leading to the premature STOP codon was present in the type I restriction endonuclease gene hsdM (SP5448_08275) within the 5448Dslo genetic backgrounds. hsdM is part of a type 1 restriction-modification system comprised of a three-gene cluster with separate restriction endonuclease (hsdR), specificity (hsdS), and methyltransferase (hsdM). The type 1 restriction-modification system has a role in DNA methylation. Investigations by others on the role of this system in gene expression and virulence determined that the type I restriction-modification system has an effect in protection against exogenous DNA (30), and that mutations (spontaneous or introduced) to hsdM within this system can be exploited to increase transformability in a strain, with no obvious change in growth or off-target gene expression (31). The outcomes of these studies suggest that the hsdM mutation in 5448Dslo was positively selected for during the transformation process and this mutation was then maintained in the passaged derivative 5448Dslo covS1. A study by Finn et al. (30) also suggests that this mutation has had no impact on off-target gene expression.
Overall, this work demonstrates that upregulation of covR/S mutation-mediated virulence factor expression is not sufficient to overcome virulence attenuation due to the absence of slo. These studies also highlight the role of DCs and neutrophils in the interaction with Strep A during infection and suggest that slo expression influences Strep A pathogenesis. SLO is secreted by nearly all Strep A isolates and the findings from this study will therefore have far-reaching implications. Taken together, these data underscore the hypothesis that covR/S mutation-mediated virulence is dictated, in part, by slo.

MATERIALS AND METHODS
Strep A strains and growth conditions. M1T1 clone 5448 and 5448AP have been previously described (4). Victor Nizet kindly provided 5448Dslo (15). All 5448Dslo covR/S mutant derivatives were SLO activity assay. The SLO activity assay was adapted from previous methods (41,42). Stationaryphase Strep A cultures (normalized to OD 600 = 1.0) were centrifuged for 10 min at 845 Â g. Dithiothreitol was added to 0.2 mm-filtered supernatants to a final concentration of 4 mM and incubated for 10 min at room temperature. Supernatants were serially diluted 1:2 in phosphate-buffered saline (PBS) in a 96-well plate. Sheep red blood cells (SRBC, 2% vol/vol, Innovative Research) were added to all wells and incubated for 30 min at 37°C 1 5% CO 2 . The plate was centrifuged for 10 min at 845 Â g, and hemoglobin release in the supernatant was measured at 450 nm. Triton X-100 (1% vol/vol in PBS, Thermo Fisher Scientific, Australia) and PBS alone were mixed with erythrocytes for the positive and negative controls, respectively. Cholesterol (25 mg/mL; Sigma-Aldrich, Australia) was added to Strep A culture supernatant and erythrocytes as a SLO-specific inhibitor, whereas trypan blue (50 mg/mL; Gibco, Australia) was used as an SLS-specific inhibitor. The number of hemolytic units/mL corresponded to the reciprocal of the dilution of supernatant that yielded 50% lysis of the erythrocyte suspension, where 100% corresponds to that caused by 1% Triton X-100 (Thermo Fisher Scientific, Australia).
For DC activation and maturation studies, dendritic cells were harvested from culture and incubated with Strep A at an MOI of 2:1 or 10:1 (Strep A:DC). Lipopolysaccharide (2 mg/mL, Sigma-Aldrich, Australia) was used as a positive control, with DCs in medium alone used as the negative control. DC/ Strep A cultures were incubated without antibiotics for 12 h at 37°C 1 5% CO 2 . Cells were collected via centrifugation and supernatants were stored at 280°C for cytokine analyses. Cells were resuspended in Fc block (CD16/CD32; BD Pharminogen, Australia) and incubated on ice for 10 min to prevent nonspecific binding of antibodies. The LIVE/DEAD Fixable Cell Stain (Life Technologies, Australia) was used according to manufacturer's instructions. For DC maturation assessment, fluorochrome-conjugated antibodies were added in a 1:100 dilution in PBS 1 1% bovine serum albumin and incubated on ice for 40 min protected from light. The antibody cocktail used included anti-mouse/rat major histocompatibility complex (MHC) II (I-Ek) fluorescein isothiocyanate (Affymetrix eBioscience), rat anti-mouse CD86 (GL-1) PE (BD Pharminogen, Australia), and hamster anti-mouse CD80 APC (16-10A1; BD Pharminogen, Australia) with the appropriate isotype controls. Samples were analyzed using a CyAn ADP flow cytometer (Beckman Coulter) employing Summit software v4.3 (Beckman Coulter).
Cytometric bead array. Cytometric bead arrays (CBA; Becton, Dickinson and Co.) were performed to quantify secreted cytokines. Each CBA was used as per manufacturer's instructions. All samples and standards were run on a CyAn ADP flow cytometer (Beckman Coulter). Cytokine quantifications were calculated using FCAP Array software v3.0 (Becton, Dickinson and Co.).
Gene expression analysis using RT-PCR. RNA was extracted from mid-log-phase cultures (OD 600 = 0.4) using the phenol-chloroform extraction method detailed in the TRIzol Plus RNA Purification kit and then purified as per the manufacturer's instructions. (Invitrogen, Australia) RNA samples were treated with RNase-free DNase I (Roche, Australia) for 20 min at 37°C, and the reaction was stopped with the addition of 0.2 M EDTA (pH 8.0) to a final concentration of 8 mM. cDNA was synthesized from 75 ng RNA using iScript Reverse Transcription Supermix for RT-PCR (Bio-Rad, Hercules, CA, USA). RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad) incorporating 0.2 mM of each primer (primer sequences provided in Table S3). The thermal profile consisted of 95°C for 3 min; 45 cycles of 95°C for 15 sec, 60°C for 1 min, and 90°C for 10 sec; and a melt curve (55°C to 95°C with 0.5°C-increments/step).
The relative amounts of gene-specific cDNA were quantified using the threshold cycle (DDCT) method, where gyrA amplification was used as the internal control. The fold change in transcript level was compared to that of gyrA, and all covR/S WT values were normalized to a baseline value of 1. The amplification efficiency was between 90% and 110% for each gene as determined by standard curve analysis over several dilutions.
covR/S gene sequencing. covR/S sequencing was conducted as previously described (7). Briefly, InstaGene Matrix (Bio-Rad, Australia) was used to extract genomic DNA from Strep A colonies as per the manufacturer's instructions. PCR was used to amplify the entire covR/S operon. Each PCR was prepared using a master mix containing 1Â GoTaq Mastermix (Promega, Australia), 0.2 mM of each primer (covRS 1F and covRS 12R, primers listed in Table S4), 50 ng template DNA, and sterile MilliQ H 2 O made up to a final volume of 50 mL. The thermal profile consisted of an initial denaturation step of 95°C for 2 min; 35 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min; then a final elongation step of 72°C for 5 min. Each amplicon was purified using the High Pure PCR Clean-Up Micro kit (Roche, Australia) following the manufacturer's instructions. Purified DNA was sequenced commercially at the Australian Genome Research Facility (Queensland, Australia).

SUPPLEMENTAL MATERIAL
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